Nicotine formulation

ABSTRACT

A method of manufacturing a nicotine-containing mucoadhesive film, by preparing an aqueous solution at a pH of from 9.5 to 13, said solution comprising (i) a nicotine salt, (ii) an alkaline pH-regulating agent, and (iii) a film-forming agent comprising an alginate salt of monovalent cation or a mixture of alginate salts of monovalent cations, the film-forming agent having a mean guluronate (G) content of from 50 to 85% by weight, a mean mannuronate (M) content of from 15 to 50% by weight, a mean molecular weight of from 30,000 g/mol to 90,000 g/mol and being such that a 10% aqueous solution thereof at a temperature of 20° C. has a viscosity of 100-1000 mPas, as measured at a shear rate of 20 rpm by use of a Brookfield viscometer with a spindle No. 2; distributing the solution onto a solid surface; and permitting the solution to dry on said surface. A nicotine-containing mucoadhesive film.

This application is a continuation of U.S. application Ser. No.16/197,954 filed Nov. 21, 2018, which is a continuation in part of U.S.application Ser. No. 14/386,083 filed Sep. 18, 2014, which is a 371 ofinternational application PCT/EP2013/0055456 filed Mar. 15, 2013 andpublished in the English language, which claims priority to Europeanapplication EP 1261483.8 filed Mar. 27, 2012 and claims the benefit ofU.S. Provisional Application No. 61/615,997 filed Mar. 27, 2012, all ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a nicotine film formulation foradministration of nicotine to a human subject and to a method forpreparing such formulation.

BACKGROUND OF THE INVENTION

A well-known therapeutic approach to aid in smoking cessation is toprovide the smoker with nicotine from sources other than cigarettes. Forexample, there are a number of commercially available nicotinereplacement products that deliver nicotine to the systemic circulationvia absorption through mucosal membranes or through the skin. Theseinclude e.g. nicotine-containing chewing gums and lozenges, as well astransdermal patches.

Both the nicotine lozenge and the nicotine chewing gum contain nicotinebitartrate or nicotine resinate. On chewing the gum or sucking thelozenge, the nicotine salt is released from the gum or lozenge andabsorbed through the lining of the mouth. However, some of the nicotinealso will be swallowed together with the saliva, which will reduce theamount entering the systemic circulation directly without passingthrough the gastric system. Another disadvantage of the lozenge orchewing gum is that the required chewing or sucking must be performedfor some time in order for the entire dose to be released, which in somecircumstances may be awkward or socially unacceptable. There are otherevident disadvantages of these forms of administration, e.g. the tastewhich is not always perceived as agreeable, the litter resulting fromthe chewed chewing gum and even the suggested possibility that the resinof the chewing gum may lead to cancer in the mouth or throat.

By the above-mentioned nicotine delivery devices absorption occurs quiteslowly and provides a low, steady-state blood level of nicotine to thepatient without the early nicotine concentration spike that occurs dueto immediate, arterial delivery of nicotine to the brain obtained whensmoking a cigarette. In fact, a goal of these therapies is to eliminatethe immediate, pleasurable effects associated with smoking while stillalleviating the nicotine withdrawal effects until complete cessation ofnicotine is physically and psychologically possible for the patient.However, this complete lack of “rush effect” experienced by the patient,may in part explain the rather low success rates of these conventionaltherapies, as discussed in e.g. U.S. Pat. No. 5,298,257 to Bannon et al.

Nicotine, or 3-[(2S)-1-methylpyrrolidin-2-yl]pyridine, is a hygroscopic,water-miscible, oily liquid alkaloid containing two basicnitrogen-containing rings, the pyrrolidine ring and the pyridine ring.

The pyrrolidine nitrogen is more basic than the pyridine nitrogen and,thus, nicotine may be dipronotated, monoprotonated or free base (i.e.unprotonated).

It is well-known that it is the free base nicotine, present in thecigarette smoke, that is behind the more rapid rush effect. However, infree base form, nicotine evaporates even at a temperature as low as roomtemperature. Furthermore, as a free base, it also is easily degraded byoxygen and light.

Free base nicotine also is very aggressive towards its environment andmigrates through most known materials. Furthermore, free base nicotine,being very hygroscopic, is very sensitive to moisture. Finally, whenexposed to oxygen or air free base nicotine turns brown. These stabilityand migration problems inherent to the free base nicotine molecule arediscussed in U.S. Pat. No. 6,790,496, which suggests a solution to thisproblem based on packaging of the nicotine-containing products inspecial packing materials.

To the knowledge of the inventors, presently, the only commerciallyavailable, non-tobacco containing products offering a rush effectsimilar to that obtained when smoking a cigarette are aerosol devices ofthe type sold under the trade mark NICORETTE® QuickMist. However, theuse of nicotine spray is not without drawbacks. For example, nicotinespray users have reported discomforts such as tingling lips, hiccups anddisagreeable taste of the aerosol product. Other drawbacks are e.g. theinherent risk of loss to the surrounding air when using the spray, andthe environmentally detrimental need for packaging material in the spraycanisters.

From the above it appears that there still is a need for a smokingcessation product that at the same time is capable of releasing freebase nicotine in a way that more or less emulates cigarettes, and stillis stable against evaporation or degradation of the nicotine.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a product fornicotine delivery that essentially solves the above-mentioned problemsof the prior art nicotine delivery products.

One important object of the present invention is to provide a nicotineformulation capable of keeping its nicotine content during storage, yetbeing able to deliver nicotine free base when administered to a humansubject.

Thus, according to a first aspect a nicotine containing mucoadhesivefilm is provided, obtainable by

-   -   preparing an aqueous solution at a pH of from 9.5 to 13, said        solution comprising

(i) a nicotine salt,

(ii) an alkaline pH-regulating agent, and

(iii) a film-forming agent comprising an alginate salt of monovalentcation or a mixture of alginate salts of monovalent cations, thefilm-forming agent having a mean guluronate (G) content of from 50 to85% by weight, a mean mannuronate (M) content of from 15 to 50% byweight, a mean molecular weight of from 30,000 g/mol to 90,000 g/mol andbeing such that a 10% aqueous solution thereof at a temperature of 20°C. has a viscosity of 100-1000 mPas, as measured at a shear rate of 20rpm by use of a Brookfield viscometer with a spindle No. 2;

-   -   distributing the solution onto a solid surface; and    -   permitting the solution to dry on said surface.

According to another aspect, there is provided a method of manufacturinga nicotine-containing mucoadhesive film, comprising:

-   -   preparing an aqueous solution at a pH of from 9.5 to 13, said        solution comprising

(i) a nicotine salt,

(ii) an alkaline pH-regulating agent, and

(iii) a film-forming agent comprising an alginate salt of monovalentcation or a mixture of alginate salts of monovalent cations, thefilm-forming agent having a mean guluronate (G) content of from 50 to85% by weight, a mean mannuronate (M) content of from 15 to 50% byweight, a mean molecular weight of from 30,000 g/mol to 90,000 g/mol andbeing such that a 10% aqueous solution thereof at a temperature of 20°C. has a viscosity of 100-1000 mPas, as measured at a shear rate of 20rpm by use of a Brookfield viscometer with a spindle No. 2;

-   -   distributing the solution onto a solid surface; and    -   permitting the solution to dry on said surface.

The film of the invention is useful for buccal transmucosal delivery ofnicotine.

Some advantages of the methods and formulations of the present inventionare that:

-   -   pH buffering systems can be omitted from the formulation of the        invention;    -   high systemic uptake of nicotine free base is obtained through        the oral mucosa;    -   the dry formulation contains only very small amounts of free        base nicotine available to reactions induced by air and light,        keeping the product stable over time;

and

-   -   nicotine is incorporated in the formulation in the form of water        soluble salt, and as such is easy to distribute evenly        throughout a solution, thereby providing a homogeneous        distribution of nicotine in the formulation.

The nicotine film of the present invention suitably is a stand-alone,one layer film.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing nicotine concentration (mean+/−SEM), in ng/ml,in blood samples collected from 5 healthy subjects over a 2-hour periodafter administration of a film dosage unit according to the inventioncontaining 2 mg of nicotine (in the form of nicotine salt).

DETAILED DESCRIPTION OF THE INVENTION

The nicotine film of the present invention is obtainable by:

-   -   preparing an aqueous solution at a pH of from 9.5 to 13, said        solution comprising

(i) a nicotine salt,

(ii) an alkaline pH-regulating agent, and

(iii) a film-forming agent comprising an alginate salt of monovalentcation or a mixture of alginate salts of monovalent cations, thefilm-forming agent having a mean guluronate (G) content of from 50 to85% by weight, a mean mannuronate (M) content of from 15 to 50% byweight, a mean molecular weight of from 30,000 g/mol to 90,000 g/mol andbeing such that a 10% aqueous solution thereof at a temperature of 20°C. has a viscosity of 100-1000 mPas, as measured at a shear rate of 20rpm by use of a Brookfield viscometer with a spindle No. 2;

-   -   distributing the solution onto a solid surface; and    -   permitting the solution to dry on said surface.

The nicotine salt may be any pharmaceutically acceptable nicotine salt.Nicotine is able to form salts with many metals and acids. The acidsthat may be used to prepare the pharmaceutically acceptable acid saltsof nicotine are those that form non-toxic acid salts, i.e., saltscontaining pharmaceutically acceptable anions, such as hydrochloride,hydrobromide, hydroiodide, nitrate, sulphate or bisulphate, succinate,maleate, fumarate, bitartrate, gluconate, saccharate, benzoate,methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate, camphorate and pamoate salts. Particularly preferred are thetartrate and bitartrate salts.

Preferably, the alkaline pH regulating agent is a strong base, such asLiOH, NaOH or KOH. In one embodiment, the alkaline pH regulating isNaOH.

The nicotine formulation of the invention additionally may comprise anysuitable excipient, such as one or more fillers or plasticizers. Anexample of a filler e.g. is microcrystalline cellulose. The plasticizer,when present, may be selected from e.g. polyethylene glycols, glyceroland sorbitol.

Optionally, the nicotine formulation of the invention also may compriseany physiologically (e.g. non-toxic at the added level) and/orpharmacologically acceptable additive, such as one or more flavouringagents (taste maskers) and/or colouring agents. Examples of flavouringagents are sorbitol, peppermint, orange flavouring, lemon flavouring,cherry flavouring, and cranberry extract. Examples of colouring agentsare titanium dioxide and green or red food colour.

The film-forming agent of the present invention is an alginate salt of amonovalent cation or a mixture of alginate salts of monovalent cations,the film-forming agent having a mean guluronate (G) content of from 50%to 85% by weight, a mean mannuronate (M) content of from 15% to 50% byweight, a mean molecular weight of from 30,000 g/mol to 90,000 g/mol andbeing such that an aqueous solution of 10% thereof at a temperature of20° C. has a viscosity of 100 mPas to 1000 mPas, as measured at a shearrate of 20 rpm by use of a Brookfield viscometer with a spindle No. 2.Such a film-forming agent is described in patent applicationPCT/SE2006/050626 (WO 2007/073346) the disclosure of which isincorporated herein by reference.

Thus, the nicotine formulation of the invention is a water-soluble film,such as a mucoadhesive film, which on application to oral mucosa adheresthereto and dissolves, allowing active ingredients contained in the filmto penetrate the mucosal membrane and enter the blood stream. Such amucoadhesive film is generally described in PCT/5E2006/050626 (WO2007/073346).

In some embodiments of a nicotine film formulation, sorbitol and/orglycerol are used as plasticizers. A suitable amount of plasticizer ise.g. from 10 to 85 g, or from 30 to 70 g, e.g. from 50 to 60 g ofplasticizer per 100 g of film-forming agent, e.g. alginate.

In some embodiments of the nicotine film formulation, filler(s) arepresent in an amount of 0-20%, e.g. 5-10% by weight of the totalpharmaceutical composition.

The nicotine formulation according to the present invention is a dryformulation, and is prepared by a method comprising a drying step. By“dry” is meant that the formulation may at most have a humiditycorresponding to equilibrium with a surrounding atmosphere having arelative humidity of from 10 to 40%, e.g. from 20 to 30%, at 25° C.

In one embodiment of the invention, the alkaline nicotine-containingfilm-forming solution (the “casting solution”) is obtained by:

-   -   preparing an aqueous solution of a nicotine salt, and an        alkaline pH regulating agent providing an alkaline pH in the        aqueous solution; and    -   admixing said aqueous solution with a film-forming agent        comprising an alginate salt of monovalent cation or a mixture of        alginate salts of monovalent cations, the film-forming agent        having a mean guluronate (G) content of from 50 to 85% by        weight, a mean mannuronate (M) content of from 15 to 50% by        weight, a mean molecular weight of from 30,000 g/mol to 90,000        g/mol and being such that a 10% aqueous solution thereof at a        temperature of 20° C. has a viscosity of 100-1000 mPas, as        measured at a shear rate of 20 rpm by use of a Brookfield        viscometer with a spindle No. 2, so as to obtain an aqueous        solution containing the film forming agent and the nicotine        salt, said solution having a pH of from 9.5 to 13.

In this embodiment, the alkaline pH regulating agent may be added to theaqueous nicotine salt solution in an amount providing a pH of at least10, or at least 11, at least 11.5, at least 12, at least 12.4, or atleast 12.5, e.g. a pH in the range of from 10 to 13, or from 11 to 13,such as from 11.5 to 13, e.g. from 12 to 13, or from 12.4 to 12.8, e.g.from 12.5 to 12.7.

In particular, the alkaline pH regulating agent is added to the aqueousnicotine salt solution in an amount such that after admixing thealkaline nicotine-containing solution (optionally containing also otheringredients, such as flavor, plasticizer, filler etc.) with the alginatesalt, a casting solution is obtained having a pH of at least 9.5, atleast 9.7, at least 10, at least 10.5, at least 10.7, at least 11, or atleast 11.5. For example, the pH_(ii) may be from 9.5 to 12.5, or from9.7 to 12.2, or from 10 to 11.7, such as from 10.5 to 11.5, e.g. from10.7 to 11.5, or from 11 to 11.5, e.g. from 11.2 to 11.5.

For example, in one embodiment, a nicotine salt such as nicotinebitartrate is mixed with water and e.g. a suitable metal ion hydroxidesalt, such as NaOH, as a basifying agent, so as to provide an aqueousnicotine solution having a pH as indicated herein above, e.g. in therange of from 10 to 13, or from 11 to 13, such as from 11.5 to 13, e.g.from 12 to 13, or from 12.4 to 12.8, e.g. from 12.5 to 12.7. In light ofthe present disclosure and by consulting also the Examples herein, theperson of ordinary skill in the art will be well enabled to find thespecific p, necessary in order to obtain the selected pH of at least 9.5for the casting solution.

Optional ingredients, such as flavor, plasticizer, filler, may be addedat any moment, e.g. after adding the basifying agent, but preferablyshould be added before admixing of the alginate. Next, the film-formingalginate is admixed with the aqueous nicotine solution and the mixturemay then be poured into suitable casting dies or onto a solid castingsurface and allowed to dry.

In one embodiment, the method for preparing the mucoadhesivenicotine-containing film of the invention comprises:

(i) preparing an aqueous solution of a nicotine salt and an alkalinepH-regulating agent, said solution having a pH_(i), which is higher thanpH_(ii);

(ii) adding, to the solution obtained in (i), a film-forming agentcomprising an alginate salt of monovalent cation or a mixture ofalginate salts of monovalent cations, the film-forming agent having amean guluronate (G) content of from 50 to 85% by weight, a meanmannuronate (M) content of from 15 to 50% by weight, a mean molecularweight of from 30,000 g/mol to 90,000 g/mol and being such that a 10%aqueous solution thereof at a temperature of 20° C. has a viscosity of100-1000 mPas, as measured at a shear rate of 20 rpm by use of aBrookfield viscometer with a spindle No. 2; so as to obtain an aqueoussolution containing the film forming agent and the nicotine salt, saidsolution having a pH_(ii) of from 9.5 to 13;

(iii) distributing the solution onto a solid surface; and

(iv) permitting the solution to dry on said surface.

In another embodiment, the method for preparing the mucoadhesivenicotine-containing film of the invention comprises

(i) preparing separately:

(a) an aqueous solution of a film-forming agent comprising an alginatesalt of monovalent cation or a mixture of alginate salts of monovalentcations, the film-forming agent having a mean guluronate (G) content offrom 50 to 85% by weight, a mean mannuronate (M) content of from 15 to50% by weight, a mean molecular weight of from 30,000 g/mol to 90,000g/mol and being such that a 10% aqueous solution thereof at atemperature of 20° C. has a viscosity of 100-1000 mPas, as measured at ashear rate of 20 rpm by use of a Brookfield viscometer with a spindleNo. 2; and

(b) an aqueous solution of a nicotine salt;

whereby at least one of the two solutions (a) and (b) contains analkaline pH regulating agent;

(ii) admixing the two solutions so as to obtain a nicotine-containingfilm-forming solution having a pH of from 9.5 to 13;

(iii) distributing the solution onto a solid surface; and

(iv) permitting the solution to dry on said surface.

In one embodiment, solution (a) contains an alkaline pH regulatingagent.

In one embodiment, solution (b) contains an alkaline pH regulatingagent.

Drying preferably is effected until the formulation reaches a level ofdryness equal to that which it would have in equilibrium with asurrounding atmosphere having a relative humidity of 10 to 40% at 25°C., e.g. 20 to 30% at 25° C., e.g. a water content of about 8% byweight.

To prepare a dry film, the process for preparing a dry film as generallydescribed in WO 2007/073346 may be followed.

For example, the casting solution is distributed onto a solid, flatsurface as a wet film having a thickness of from e.g. 0.1 to 4 mm, suchas 0.2 to 2 mm, e.g. 0.5 to 1.5 mm. The wet film then is allowed to dryon the surface, e.g. in room temperature or in a ventilated oven ordrying cabinet at a temperature of 45-60° C., e.g. at a temperature offrom 52 to 54° C., for a time period of e.g. 20 to 40 minutes, or from20 to 30 minutes.

After drying at least partly of the film, the dry or semi-dry film thusobtained may be divided into suitably sized dosage units, e.g. bycutting or punching.

The film may be imprinted imprinted at one or both sides with words,figures or other markings, e.g. a trade mark or an indicating of thedosage, using an ink suitable for human ingestion. For example a 2 mgdosage unit may be imprinted with “2 mg”.

The dry dosage units may be packaged into suitable containers, e.g.resealable containers of a water and air tight material suitable for usein packaging of products for human ingestion, e.g. a metallisedpolyethylene film (Alu/PET).

As illustrated in the stability test described herein below, by themethod of the present invention, a nicotine film formulation having ahigh shelf life is obtained. It is remarkable that the nicotine contentof the film remains essentially unchanged over a time period of nearlyfour months without any precautions being taken to preserve the filmfrom either light or surrounding air.

When administered to the mouth of a human subject, the formulation willdissolve by the action of the saliva, releasing the basifying pHregulating agent and nicotine free base. As the nicotine free basepenetrates the oral lining and enters the blood stream, the desired rusheffect may be obtained.

It is an advantageous feature of the nicotine formulation of the presentinvention that it is in the form of a mucoadhesive film. When applied tothe mucous membrane of the mouth, the dry film will adhere thereto anddissolve over a given time period, e.g. 1 minute to 10 minutes, such as1-5 minutes, or 1-3 minutes. As the film dissolves, the nicotinic saltand the basifying agent are released. The basifying agent will provide ahigh local pH in the liquid phase formed by the saliva at the oralmucosa in contact with the dissolving mucoadhesive film. In this high pHliquid solution nicotine will be present again as a free base, and assuch will penetrate the mucous membrane and enter the blood stream ofthe body. Systemic parenteral delivery of free base nicotine thereby isobtained.

The dry film formulation according to the invention preferably has athickness of 0.01 to 2 mm, or 0.02 to 1 mm, e.g. 0.05 to 0.5 mm, or from0.06 to 0.4 mm, or from 0.06 to 0.1 mm, e.g. about 0.07 mm

In one embodiment, the nicotine-containing film of the invention isprovided in dosage units. Such dosage unit may be of any suitablesurface area, having regard to the concentration of the nicotine saltwithin the film and the suitable nicotine dosage to be administered. Asan example, a dosage unit having a surface area of from 1 cm² to 10 cm²may be selected, e.g. from 2 to 8 cm², or from 4 to 7 cm², such as about6 cm². It will be within the knowledge of the skilled person to adaptthe size and shape of the film dosage unit having regard to suchparameters as e.g. the loading of the nicotine salt within the film andthe required dosage. Also, it should be realized that the film dosageunit may have any appropriate shape, e.g. it may be rectangular,circular, oblong, oval etc.

A suitable dosage unit e.g. may be contain from 0.5 mg to 4 mg nicotinein the form of nicotine salt, e.g. from 1 to 2 mg nicotine, or any othersuitable amount. For example, a dosage unit may be a dry film unithaving a surface area of 3 cm², a thickness of about 0.2 mm andcontaining about 2 mg nicotine. In one embodiment, the dosage unit is adry film unit having a surface area of 6 cm², a thickness of about 0.07mm, containing about 2 mg nicotine. In another embodiment, the dosageunit is a dry film unit having a surface area of 6 cm², a thickness ofabout 0.07 mm, containing about 1 mg nicotine.

An important advantageous feature of the nicotine film of the presentinvention is its capacity of providing a high systemic availability dueto the transmucosal absorption of nicotine.

Compared to nicotine chewing gums, where a large part of the nicotine isswallowed down with the saliva, this will allow for a reduced dosage ofnicotine.

A further advantage provided by the nicotine film of the invention isthe very simple, easy to handle dosage form, compared e.g. to theaerosol spray.

Another advantageous feature of the nicotine film of the invention isstorage stability of the nicotine in the film, which may allow for amulti-dosage package of dosage units of the film without any need forindividual packaging of each dosage unit. Thus, in one embodiment, aresealable package is provided containing a plurality ofnicotine-containing film dosage units according to the invention. Forexample, such a resealable package may contain from 5 to 200 dosageunits, or from 10 to 100 dosage units, e.g. from 20 to 50 dosage units,such as 30 dosage units.

In some embodiments, each dosage unit is packaged separately in an airand water tight material, such as a metallised polymeric film, e.g. anAlu/PET film. For example, each dosage unit may be provided separatelyin an Alu/PET envelope.

Herein below, the invention will be illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

A mucoadhesive nicotine-containing film according to the invention wasprepared using the ingredients listed in Table 1.

TABLE 1 Ingredient Amount nicotine tartrate 3.7 g water 174.5 mlsorbitol   6 g glycerol   6 g NaOH 2M  20.5 ml titanium dioxide 0.3 glemon flavour    2 ml peppermint flavour    1 ml sodium alginate(Protanal ® LFR 5/60, 26.7 g  sold by FMC BioPolymer)

The film was prepared as follows: In a beaker, water was mixed withnicotine tartrate and NaOH until a clear solution was obtained. The pHwas adjusted to within a range of from 11.8 to 12.8. Titanium dioxidewas added and the solution was sonicated to provide a homogenousdispersion of titanium dioxide in the nicotine solution. Next, ⅓ of thealginate was added and the solution was mixed in a mixer so as to obtaina visibly homogeneous liquid phase. While maintaining the stirring,glycerol, sorbitol and the flavouring agents were added. The remainderof the alginate then was added and the mixing was continued untilobtaining a homogenous, viscous liquid phase. The liquid mixture thenwas transferred to a glass beaker and sonicated again to remove any airbubbles therein. Subsequently, ¼ of the liquid mixture was distributedhomogeneously over a glass plate at a thickness of 0.89 mm by means of adraw down blade for wet film application. The film was dried in a dryingcabinet at a temperature of 45 to 60° C. for 25 minutes. The dry filmwas cut into rectangular pieces of 2×3 cm² and the samples of nicotinefilm were placed in clean, plastic pockets.

A stability study of nicotine in the film of the invention was performedin order to establish the shelf life of nicotine in an opened multi-dosepackage. The dosage units prepared in EXAMPLE 1 were used in the test.At day 0 of the study, the plastic pocket containing the dry filmsamples was opened and the nicotine concentration in 3 dosage units(i.e. three 6 cm² pieces) of the film formulation was determined. Theremaining dosage units contained in the plastic pocket were stored inthe open at a temperature ranging from 21 to 24° C. and a relativehumidity of 19-32%. These film dosage units were not stored in the dark,but simply kept in the open, on a shelf in the laboratory. The nicotineconcentration of 3 different dosage units was measured again at day 7and at day 102, respectively. The results are listed in Table 2 hereinbelow.

TABLE 2 Days after opening of package Nicotine mg/dosage unit 0 1.328 71.328 102 1.310

Overview of method and equipment for analysis

Analysis was performed using the following equipment:

Isocratic HPLC-pump: Constametric Model III

Auto injector: Dynamax Model AI3 (Loop volume 50 μl)

Column heater: Jones Chromatography Model 7981 (Temperature 35° C.)

Column: Dr Maisch, Reprosil-Pur Basic, C18-AQ 5 μm, 150×4 mm

UV-detector: PerSeptive Biosystems UVIS-205 (at 260 nm)

Flow rate 1 ml/min

Mobile phase: 30% ACN, 70% 10 mM Phosphate Buffer pH 8.5

Diluent 15% ACN, 85% 10 mM Phosphate Buffer pH 8.5

A standard curve using nicotine bitartrate dihydrate in diluent wasused. The samples where diluted in 100 ml of diluent and filtratedthrough a 0.4 μm filter.

Example 2

A mucoadhesive nicotine-containing film according to the invention wasprepared essentially as described in EXAMPLE 1, using the ingredientslisted in Table 3.

TABLE 3 Ingredient Amount nicotine tartrate 5.1 g water 171 ml sorbitol  7 g glycerol   7 g NaOH 2M  24 ml titanium dioxide 0.3 g lemon flavour 2 ml peppermint flavour  1 ml sodium alginate (Protanal ® LFR 5/60,26.7 g  sold by FMC BioPolymer)

Dosage units containing 2 mg nicotine/unit were prepared. The systemicdelivery of nicotine by peroral administration of these dosage units wasassessed on 5 healthy subjects.

Before administration of the dosage unit, a blood sample was withdrawnfrom the subject to establish a zero level. At that point of time, thesubjects had not used any nicotine-containing product for at least 24hours.

At time zero, a film dosage unit of the invention was applied to thepalate of each subject. Blood samples were collected from each subjectat regular intervals during 2 hours. Plasma was separated, frozen usingdry ice and sent to a GLP accredited laboratory for analysis. Theanalytical method was developed at the laboratory and validatedaccording to the FDA Guidance for Industry—Bioanalytical MethodValidation (CDER, May 2001). The tabulated summary of this validation isshown in Table 4.

TABLE 4 Validation summary for the quantification of nicotine in humanplasma by LC-MS/MS Analyte Nicotine Internal standard Nicotine-d4 Matrix(Anticoagulant) Human Plasma (Lithium Heparin) SOP Number 5-85Analytical Method High performance liquid chromatography (HPLC) coupledto tandem mass spectrometry with multiple reaction monitoring (MRM)Detector AB/MDS Sciex API 4000 Human Plasma Volume Required 100 μLStandard Curve Range 0.5-100 ng/mL QC concentrations 1.5, 15, 80 ng/mLRegression Type Linear regression (weighted 1/concentration 2)Quantification Method Peak area ratio Selectivity No interfering peaksnoted in blank plasma samples LLOQ Validation Samples Precision (%) Bias(%) Inter-batch 7.9% −4.2% Intra-batch 4.05% to 6.81% −0.91% to 12.66%Quality Control Samples Precision (%) Bias (%) Inter-batch Low 7.3% 2.3%Inter-batch Medium 2.0% 2.9% Inter-batch High 2.4% 5.6% Intra-batch Low3.19% to 3.55% −6.96% to 7.03%   Intra-batch Medium 0.96% to 2.52% 1.39%to 3.91% Intra-batch High 1.83% to 2.50% 3.84% to 6.68% RecoveryRecovery (%) Analyte Low 99% Analyte Medium 134%  Analyte High 78%Internal Standard 90% Long-term Stability 103 days at −20° C. Short-termStability 24 hours at room temperature and 120 hours at 4° C.Freeze-Thaw Stability 4 cycles at −20° C. Stock Solution Stability 29days in acidified water at 4° C. Processed Sample Stability 72 hours at4° C. and room temperature

Samples of human plasma with an added internal standard were extractedusing a liquid-liquid extraction procedure. After evaporation andresuspension in LC mobile phase, the samples were analyzed by LC-MS/MS.Positive ions were monitored in the multiple reaction monitoring (MRM)mode. Quantification was by peak area ratio.

The results, in terms of plasma nicotine concentration (in ng/ml) in theblood plasma samples, are illustrated in FIG. 1. The indicated valuesare mean values calculated from the 5 subjects participating in thetest.

Example 3

The ingredients used in the Example are indicated in Table 5.

TABLE 5 Ingredient Amount nicotine bitartrate 5.1 g sorbitol   7 gglycerol   7 g NaOH 2M enough to pH_(i) (V₁ ml) water enough to 195 mlwith V₁ and V₂ titanium dioxide 0.3 g lemon flavour 2 ml peppermintflavour 1 ml sodium alginate (Protanal ® LFR 5/60, 26.7 g  sold by FMCBioPolymer)

An aqueous nicotine bitartrate solution of 5.1 g nicotine bitartrate inabout 160 ml of water was prepared and 2 M NaOH was added until analkaline pH was reached (pH_(i)). Titanium dioxide, dissolved in a smallamount of water (V₂ ml), was added. The total volume of the aqueousalkaline solution was adjusted to 195 ml by addition of further water.Sorbitol, glycerol, and flavours were added to the alkaline solution,followed by the sodium alginate. The pH was measured (pH_(ii)) on asample diluted 1:2. The solution then was cast and dried to provide adry film. From the dry film 6 cm², 0.07 mm thick samples weighing 70 mgwere cut and dissolved in 10 ml of water, and the pH of the aqueoussolution was measured.

The nicotine contents of film samples were measured directly afterdrying and after 30 days storage at 25° C., ambient RH, packed inAlu/PET pouches. The results are shown in Table 6.

TABLE 6 pH_(i) in nicotine pH_(ii) in pH dried nicotine content solutionbefore wet cast film in drop from addition of alginate (50/50 H₂O) H₂O(10 mL) initial value (%) 10.7 8.8  8.47 >5% 12.22  9.24  8.81 >5% 12.379.5  9.04 <5% 12.52 11.18  9.82 <5% 12.88 12.3  10.41 <5%

From the results in Table 6, it surprisingly appears that a nicotinecontaining film prepared by use of a film-forming solution of theinvention, having a pH of at least 9.5, has a good stability, whereaswhen the pH of the film-forming solution is lower than about 9.5, thestability of the nicotine in the dry film is insufficient.

Example 4

The ingredients used in Example 4 are listed in Table 7.

TABLE 7 Ingredient Amount nicotine bitartrate  5.1 g sorbitol (70%)    7g glycerol (85%)    7 g NaOH 2M  24 ml water 191 ml titanium dioxide 0.3 g lemon oil  2 ml peppermint flavour  1 ml sodium alginate(Protanal ® LFR 5/60, 23.76 g sold by FMC BioPolymer) sodium alginate(Protanal ® LFR 10/60,  2.94 g sold by FMC BioPolymer)

An aqueous dispersion of titanium dioxide was prepared. Nicotinebitartrate was admixed with the dispersion and dissolved therein, andthen NaOH was added. Sorbitol, glycerol, and flavours were added to thealkaline solution, followed by the sodium alginate to obtain afilm-forming solution having a pH of 11.6. The solution was cast anddried to provide a dry film. From the dry film, samples were cut havinga surface area of 3 cm², a thickness of 0.07 mm and weighing 35 mg. Thefilm samples were separately packaged in aluminium/PET/PE foil envelopesand sealed, and the packaged films were stored at room temperature.

The nicotine contents of the films were determined using HPLC (HighPressure Liquide Chromatograph), by dissolving each film in 50 ml ofbuffer (pH 8.5) and 15% acetonitrile and using a mobile phase of 30%acetonitrile and 70% pH 8.5 buffer. A nicotine standard was used to keeptrack of the response rate for the system and the response was measuredin mVs.

The first nicotine determination was made on a freshly made and driedfilm, the second was made on a film from the same batch, after 4 monthsof storage. Each time, three 35 mg film samples were used and the meanwas calculated based on the three measurements. The determined drop ofnicotine content according to the HPLC analysis was less than 5%.

Example 5

The ingredients used in Example 5 are listed in Table 8.

TABLE 8 Ingredient Amount nicotine bitartrate  5.1 g sorbitol (70%)   7g glycerol (85%)   7 g NaOH 2M  15 ml buffer 0.1M Na₂CO₃/NaHCO₃, 50/50180 ml (pH 8.5) titanium dioxide  0.3 g lemon oil  2 ml peppermintflavour  1 ml sodium alginate (Protanal ® 26.7 g LFR 5/60, sold by FMCBioPolymer)

A dispersion of titanium dioxide in buffer was prepared. Nicotinebitartrate was admixed with the dispersion and dissolved therein, andthen NaOH was added. Sorbitol, glycerol, and flavours were added to thealkaline solution, followed by sodium alginate to obtain a film-formingsolution having a pH of 10.75. The solution was cast and dried toprovide a dry film. From the dry film, samples were cut having a surfacearea of 3 cm², a thickness of 0.07 mm and weighing 35 mg.

The film samples were separately packaged in aluminium/PET/PE foilenvelopes, which were subsequently sealed and stored at roomtemperature.

The first nicotine determination was made on a freshly made and driedfilm, the second was made on a film from the same batch, after 4 monthsof storage. Each time, three 35 mg film samples were used and the meanwas calculated based on the three measurements. The determined drop ofnicotine content according to the HPLC analysis was less than 5%.

Example 6

The ingredients used in Example 6 are listed in Table 9.

TABLE 9 Ingredient Amount nicotine bitartrate  5.1 g sorbitol (70%)   7g glycerol (85%)   7 g NaOH 2M  24 ml water 171 ml titanium dioxide  0.3g lemon oil  2 ml peppermint flavour  1 ml sodium alginate (Protanal ®LFR 5/60, 26.7 g sold by FMC BioPolymer)

An aqueous dispersion of titanium dioxide was prepared. Nicotinebitartrate was admixed with the dispersion and dissolved therein, andthen NaOH was added. Sorbitol, glycerol, and flavours were added to thealkaline solution, followed by sodium alginate to obtain a film-formingsolution having a pH of 10. The solution was cast and dried to provide adry film. From the dry film, samples were cut having a surface area of 3cm², a thickness of 0.07 mm and weighing 35 mg.

The film samples were separately packaged in aluminium/PET/PE foilenvelopes, which were subsequently sealed and stored at roomtemperature.

The first nicotine determination was made on a freshly made and driedfilm, the second was made on a film from the same batch, after 4 monthsof storage. Each time, three 35 mg film samples were used and the meanwas calculated based on the three measurements. The determined drop ofnicotine content according to the HPLC analysis was less than 5%.

Example 7

The ingredients used in Example 7 are listed in Table 10.

TABLE 10 Ingredient Amount nicotine bitartrate  5.1 g sorbitol (70%)   7g glycerol (85%)   7 g NaOH 2M  25 ml water 171 ml titanium dioxide  0.3g lemon oil  2 ml peppermint flavour  1 ml sodium alginate (Protanal ®LFR 5/60, 26.7 g sold by FMC BioPolymer)

An aqueous dispersion of titanium dioxide was prepared. Nicotinebitartrate was admixed with the dispersion and dissolved therein, andthen NaOH was added. Sorbitol, glycerol, and flavours were added to thealkaline solution, followed by sodium alginate to obtain a film-formingsolution having a pH of 11. The solution was cast and dried to provide adry film. From the dry film, samples were cut having a surface area of 3cm², a thickness of 0.07 mm and weighing 35 mg.

The film samples were separately packaged in aluminium/PET/PE foilenvelopes, which were then sealed and stored at room temperature.

The first nicotine determination was made shortly after filmpreparation, the second was made after 4 months of storage. Each time,three 35 mg film samples were used and the mean was calculated based onthe three measurements. The determined drop of nicotine contentaccording to the HPLC analysis was less than 5%.

Comparative Example 1

The ingredients used in Comparative Example 1 are listed in Table 9.

TABLE 11 Ingredient Amount nicotine bitartrate  5.1 g sorbitol (70%)   7g glycerol (85%)   7 g NaOH 2M  22 ml water 171 ml titanium dioxide  0.3g lemon oil  2 ml peppermint flavour  1 ml sodium alginate (Protanal ®LFR 5/60, 26.7 g sold by FMC BioPolymer)

An aqueous dispersion of titanium dioxide was prepared. Nicotinebitartrate was admixed with the dispersion and dissolved therein, andthen NaOH was added. Sorbitol, glycerol, and flavours were added to thealkaline solution, followed by sodium alginate, to obtain a film-formingsolution having a pH of 9.3. The solution was cast and dried to providea dry film. From the dry film, samples were cut having a surface area of3 cm², a thickness of 0.07 mm and weighing 35 mg.

The film samples were separately packaged in aluminium/PET/PE foilenvelopes, which were subsequently sealed and stored at roomtemperature.

The first nicotine determination was made shortly after filmpreparation, the second was made after 4 months of storage. Each time,three 35 mg film samples were used and the mean was calculated based onthe three measurements. The determined drop of nicotine contentaccording to the HPLC analysis exceeded 7%.

Comparative Example 2

The ingredients used in Comparative Example 2 are listed in Table 12.

TABLE 12 Ingredient Amount nicotine bitartrate 5.5 g sorbitol (70%)   3g glycerol (85%)   2 g buffer 0.1M K₂HPO₄/KH₂PO₄ (pH 8.5) 80 ml sodiumalginate (Protanal ® LFR 5/60,  11 g sold by FMC BioPolymer)

Nicotine bitartrate was mixed with the buffer. Glycerol and sorbitolwere added. Sodium alginate was added to the thus prepared aqueoussolution at room temperature in small portions and mixed until ahomogenous solution was obtained, to obtain a film forming solutionhaving a pH of 5.5. A film prepared at such low pH would not be usefulfor mucosal administration of nicotine, since on dissolution in themouth, nicotine would be present only in ionized form, not in thebioavailable free base form.

1. A mucoadhesive nicotine-containing film, obtainable by preparing anaqueous solution at a pH of from 9.5 to 13, by admixing: (i) a nicotinesalt, (ii) an alkaline pH regulating agent, and (iii) a film-formingagent comprising an alginate salt of monovalent cation or a mixture ofalginate salts of monovalent cations, the film-forming agent having amean guluronate content of from 50 to 85% by weight, a mean mannuronatecontent of from 15 to 50% by weight, a mean molecular weight of from30,000 g/mol to 90,000 g/mol and being such that a 10% aqueous solutionthereof at a temperature of 20° C. has a viscosity of 100-1000 mPas, asmeasured at a shear rate of 20 rpm by use of a Brookfield viscometerwith a spindle No. 2; distributing the solution onto a solid surface;and permitting the solution to dry on said surface.
 2. The filmaccording to claim 1, wherein the pH regulating agent is LiOH, NaOH orKOH.
 3. The film according to claim 1, wherein the nicotine salt is anacid addition salt.
 4. The film according to claim 3, wherein the acidaddition salt is a salt of tartaric acid.
 5. The film according to claim2, wherein the nicotine salt is an acid addition salt.
 6. The filmaccording to claim 5, wherein the acid addition salt is a salt oftartaric acid.
 7. The film according to claim 1, comprising aplasticizer.
 8. The film according to claim 1, comprising a filler. 9.The film according to claim 1, having a thickness in the range of from0.01 mm to 2 mm.
 10. The film according to claim 1, having a loss ofnicotine of less than 5% by weight after 100 days in air at atemperature ranging from 21 to 24° C. and a relative humidity of 19-32%.11. A nicotine dosage unit comprising a film according to claim
 1. 12.The dosage unit according to claim 11, having a surface area in therange of from 2 to 8 cm².
 13. The dosage unit according to claim 11,having a nicotine content in the range of from 0.5 mg to 4 mg.
 14. Thedosage unit according to claim 12, having a nicotine content in therange of from 0.5 mg to 4 mg.
 15. A method of manufacturing anicotine-containing mucoadhesive film, comprising: preparing an aqueoussolution at a pH of from 9.5 to 13, by admixing: (i) a nicotine salt,(ii) an alkaline pH regulating agent, and (iii) a film-forming agentcomprising an alginate salt of monovalent cation or a mixture ofalginate salts of monovalent cations, the film-forming agent having amean guluronate content of from 50 to 85% by weight, a mean mannuronatecontent of from 15 to 50% by weight, a mean molecular weight of from30,000 g/mol to 90,000 g/mol and being such that a 10% aqueous solutionthereof at a temperature of 20° C. has a viscosity of 100-1000 mPas, asmeasured at a shear rate of 20 rpm by use of a Brookfield viscometerwith a spindle No. 2; distributing the solution onto a solid surface;and permitting the solution to dry on said surface.
 16. The methodaccording to claim 15, comprising adding a plasticizer to the solution.17. The method according to claim 15, comprising adding a filler to thesolution.
 18. The method according to claim 15, comprising dividing thefilm into dosage units.
 19. A nicotine-containing film formulation forbuccal administration of free base nicotine, wherein the filmformulation has a decrease in nicotine content of less than 5% by weightafter 100 days in air at a temperature ranging from 21 to 24° C. and arelative humidity of 19-32%.
 20. A method of preparing anicotine-containing film formulation for buccal administration of freebase nicotine, wherein the film formulation has a decrease in nicotinecontent of less than 5% by weight after 100 days in air at a temperatureranging from 21 to 24° C. and a relative humidity of 19-32%, said methodcomprising preparing an aqueous solution at a pH of from 9.5 to 13, byadmixing: (i) a nicotine salt, (ii) an alkaline pH regulating agent, and(iii) a film-forming agent comprising an alginate salt of monovalentcation or a mixture of alginate salts of monovalent cations, thefilm-forming agent having a mean guluronate content of from 50 to 85% byweight, a mean mannuronate content of from 15 to 50% by weight, a meanmolecular weight of from 30,000 g/mol to 90,000 g/mol and being suchthat a 10% aqueous solution thereof at a temperature of 20° C. has aviscosity of 100-1000 mPas, as measured at a shear rate of 20 rpm by useof a Brookfield viscometer with a spindle No. 2; distributing thesolution onto a solid surface; and permitting the solution to dry onsaid surface.